PROJECT SUMMARY The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue would represent an important advance in cancer therapy by reducing toxicity to healthy tissue. Acidity is a hallmark of the tumor microenvironment that can be targeted using pH-low insertion peptides (pHLIPs). Cargo attached to pHLIP can be selectively delivered to tumor cells in vivo based on the acidity of the tumor microenvironment. The objective of this proposal is to develop a tool to inhibit DNA repair specifically within the tumor microenvironment while sparing normal tissue, with a long-term goal of developing novel cancer treatments with wider therapeutic indices. DNA repair is crucial for tumor cell survival after exposure to ionizing radiation (IR), and thus inhibiting DNA repair causes increased cell death after IR exposure (radiosensitization). Furthermore, in susceptible genetic backgrounds, inhibition of DNA repair causes cell death in the absence of radiation through a mechanism known as synthetic lethality. Small molecule inhibitors of non-homologous end joining (NHEJ), a critically important pathway in DNA repair, cause exquisite radiosensitization. However, the clinical use of these inhibitors has been hindered by in vivo toxicity and poor bioavailability. This proposal aims to use new technologies to overcome the current limitations in targeting NHEJ by developing peptide nucleic acid (PNA) based oligomers to reduce expression of the essential NHEJ factor Ku80 and using pHLIPs to selectively deliver these PNA molecules to tumors (pHLIP-?Ku80(?)), with the hypothesis that this approach will allow for the selective radiosensitization of tumor cells. This hypothesis will be tested through the following aims. To evaluate the activity of pHLIP-?Ku80(?) against cancer cells in culture (Aim 1), pHLIP-conjugated PNAs with antisense activity against Ku80 will be tested for pH-dependent activity against cancer cells in culture. Specifically, effects on DNA repair and cell survival after IR will be determined. Efforts will also be made to identify genetic backgrounds that demonstrate synthetic lethality with pHLIP-?Ku80(?) treatment. To evaluate and optimize the tumor radiosensitizing effects of pHLIP-?Ku80(?) in vivo (Aim 2), the pharmacology and toxicity of pHLIP-?Ku80(?) treatment will be assessed, and tumor growth delay and clonogenic survival assays will be used to measure radiosensitization and synthetic lethal interactions. The development of pHLIP-?Ku80(?) would provide a means of selectively radiosensitizing tumors cells regardless of tumor type, biomarker expression, and genetic background. By inhibiting DNA repair selectively in tumors, this approach spares healthy tissue and is thus expected to reduce toxicity. Therefore, pHLIP-?Ku80(?) could be used to improve the efficacy of radiation therapy without causing additional toxicity, marking an important clinical advancement. !